1. Field of the Invention
The present invention is directed to an improved process for separating and preferably recovering individual polar protic monomers and/or oligomers, including without limitation flavan-3-ols, according to their degree of polymerization, using diol-phase liquid chromatography (LC).
It is known that individual flavan-3-ols exhibit distinct properties and have distinct applications for human and animal use. Improved separation and recovery of individual flavan-3-ols on the basis of degree of polymerization will allow for more targeted and efficacious use thereof.
2. Discussion of the Related Art
Proanthocyanidins, the oligomers and polymers of flavan-3-ols, are the second most abundant natural plant phenolics after lignin. The flavan-3-ol subunits are linked primarily through a carbon-carbon bond from the 4 position of one subunit to the 8 position of another subunit (C4→C8), and to a lesser extent through C4→C6 linkage.
Proanthocyanidins include B-type and A-type proanthocyanidins. In the B-type proanthocyanidins, the monomers are linked via C4→C6 and/or C4→C8 interflavan linkages. Oligomers with exclusively C4→C8 linkages are linear, while the presence of at least one C4→C6 linkage results in a branched oligomer. By contrast, A-type proanthocyanidins are doubly linked oligomers containing linkages at C2-O-C7 as well as at C4→C6 or C4→C8.
The molecular weight of proanthocyanidins typically is expressed as degree of polymerization (DP). Individual oligomers are commonly referred to as dimers, trimers, etc.
Procyanidins represent the largest class of proanthocyanidins. Gu et al. showed that out of 41 foods found to contain proanthocyanidins, 27 contained procyanidins. See J. Agric. and Food Chem. 51 (2003) 7513. Procyanidins may include (−)-epicatechin, (+)-epicatechin, (+)-catechin and/or (−)-catechin monomeric units, as well as gallated catechins such as (−)-catechin gallate, (+)-catechin gallate, (−) epicatechin gallate and/or (+)-epicatechin gallate.
It is known that proanthocyanidins play important roles in the color stability, astringency, and bitterness of plant foods. See, e.g., Haslam, “Practical Polyphenols: From Molecular Recognition and Physiological Action” (Cambridge U. Press, 1998. However, the notoriety of proanthocyanidins has increased due to the potential health benefits of these phenolic compounds. See, e.g., Bagchi et al., Toxicology 148 (2000) 187; Foo et al., J. Natural Products 63 (2000) 1225; and Steinberg et al., Am. J. Clin. Nutri. 77 (2003) 1466).
It is also known that individual procyanidin oligomers present specific characteristics and potential benefits for use in humans and animals. For example, U.S. Pat. No. 5,211,944 (Tempesta) discloses that procyanidin oligomers having a degree of polymerization (DP) of 2-11 possess significant antiviral activity and are useful in treating warm-blooded animals, including humans infected with paramyxovaridae such as respiratory syncytial virus, orthomyxovaridae such as influenza A, B and C, and herpes viruses such as Herpes Simplex virus. U.S. Pat. No. 5,554,645 (Romanczyk, Jr. et al.) discloses antineoplastic compositions comprising procyanidin oligomers having a 3-11 together with a suitable carrier. U.S. Pat. No. 5,891,905 (Romanezyk, Jr., et al.) discloses that procyanidin oligomers having a 5-12 are useful as antioxidants. U.S. Pat. No. 6,524,630 (Schmitz et al.) discloses the use of cocoa procyanidin oligomers having a 2-18 together with acetylsalilcylic acid as an anti-platelet therapy.
Given their structural complexity and diversity in nature, the history of proanthocyanidin analysis is rich. See Santos-Buelga et al., “Processes in Polyphenol Analysis,” Royal Society of Chemistry, Cambridge, 2003, p. 267). Lea described the use of normal-phase high performance liquid chromatography (NP-HPLC) for procyanidin analysis in J. Sci. Food and Agriculture (1979) 30:833 and also observed that using a Sephadex LH-20 column under isocratic conditions resulted in an elution order where the larger oligomers were retained longer than the smaller oligomers. See Lea et al., Am. J. Enology and Viticulture (1979) 30:289. Wilson et al. disclosed using a gradient mobile phase in connection with tetrahydrofuran-hexane-acetic/formic acid-isopropanol over a cyano column to achieve partial separation based on the degree of polymerization (DP) of apple juice procyanidins. See Sci. Food. Agric. (1981) 32:257.
Significant improvements in the separation and resolution of procyanidin oligomers have been achieved on silica stationary phases. See Rigaud et al., Chromatogr. (1993) 654:179; Cheynier et al., Processes in Enzymology (1999) 299; Natsume el al., Biosci. Biotechnol. Biochem. (2000) 64:2581. Resolution of procyanidin oligomers up to the pentamer (DP=5) has been obtained. Hammerstone et al. disclosed modifications of this process leading to improvements in resolution of monomers through decamers in the analysis of unfermented, defatted cacao beans. See J. Agric. and Food Chem. (1999) 47:490. Gu et al. disclosed still further improvements leading to the elution of a decamer (DP=10), as well as enhancement in overall peak shape and resolution. See J. Agric. and Food Chem. (2000) 50:4852.
These HPLC processes use gradient mobile phases consisting of methylene chloride-methanol-acetic/formic acid-water to achieve separation of procyanidin oligomers out to the pentamer with an alternate process for separation of apple procyanidins using hexane-methanol-ethyl acetate and/or hexane-acetone over a silica column. See Yanagida et al., J. Chromatogr. A (2000) 890:251.
However, current HPLC processes, including those of Gu et al., have several shortcomings. First, the use of chlorinated solvents such as methylene chloride (also referred to as dichloromethane) presents safety concerns. This is especially an issue when isolated fractions from the larger scale systems may be targeted for further biological study. It is known that exposure to methylene chloride affects the skin, eyes, central nervous system (CNS), and cardiovascular system, and that short-term exposure can cause fatigue, weakness, sleepiness, light-headedness, numbness of limbs, tingling skin, nausea, and irritated skin and eyes. Chronic exposure to methylene chloride has been linked to cancer of the lungs, liver, and pancreas in laboratory animals. Methylene chloride also is a mutagen that may cause birth defects if women are exposed to it during pregnancy.
Tetrahydrofuran (THF), another common HPLC solvent, is known to irritate the eyes of human subjects, as well as the mucous membranes and the gastrointestinal tract. Overexposure to THF may cause coughing, shortness of breath, dizziness, central nervous system (CNS) depression, intoxication, and collapse.
Other problems with current HPLC processes for separating procyanidin oligomers include the historical problem associated with normal phase (NP) separation of procyanidins when using silica as the stationary phase, viz., column to column variability. Frequently, oligomers have reduced peak intensities or are not detected at all, as they are thought to be adsorbed on the silica surface. The use of water in the mobile phase—as required for peak shape in the NP separation of procyanidins—further degrades column to column reproducibility. Also, from a practical standpoint, most current HPLC processes for separating individual flavan-3-ols, including procyanidin oligomers, involve the use of tertiary or quaternary mobile phases, and thus are beyond the capabilities of analytical laboratories lacking sophisticated quaternary HPLC pumps.
What is needed is an improved process for identifying the individual polar protic oligomers on the basis of DP, as well as a process for separating and recovering individual polar protic oligomers based on DP that avoids use of dangerous and environmentally hazardous solvents, that provides improved separation and recovery of individual polar protic oligomers, that is suitable for use in analytical laboratories equipped only with a customary binary HPLC pump, and that is suitable for the recovery of specific oligomers on a preparative scale.